nbqx attenuates excitotoxic injury in developing white matter

NBQX Attenuates Excitotoxic Injury in Developing White Matter

Pamela L. Follett, Paul A. Rosenberg, Joseph J. Volpe, and Frances E. Jensen

Department of Neurology and Program in Neuroscience, Children’s Hospital and Harvard Medical School, Boston,Massachusetts 02115

The excitatory neurotransmitter glutamate is released from axonsand glia under hypoxic/ischemic conditions. In vitro, oligoden-drocytes (OLs) express non-NMDA glutamate receptors (GluRs)and are susceptible to GluR-mediated excitotoxicity. We evalu-ated the role of GluR-mediated OL excitotoxicity in hypoxic/ischemic white matter injury in the developing brain. Hypoxic/ischemic white matter injury is thought to mediate periventricularleukomalacia, an age-dependent white matter lesion seen inpreterm infants and a common antecedent to cerebral palsy.Hypoxia/ischemia in rat pups at postnatal day 7 (P7) producedselective white matter lesions and OL death. Furthermore, OLs inpericallosal white matter express non-NMDA GluRs at P7. Uni-lateral carotid ligation in combination with hypoxia (6% O2 for 1

hr) resulted in selective, subcortical white matter injury with amarked ipsilateral decrease in immature and myelin basicprotein-expressing OLs that was also significantly attenuated by6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). Intra-cerebral AMPA demonstrated greater susceptibility to OL injuryat P7 than in younger or older pups, and this was attenuated bysystemic pretreatment with the AMPA antagonist NBQX. Theseresults indicate a parallel, maturation-dependent susceptibility ofimmature OLs to AMPA and hypoxia/ischemia. The protectiveefficacy of NBQX suggests a role for glutamate receptor-mediated excitotoxic OL injury in immature white matter in vivo.

Key words: white matter; glutamate receptors; AMPA; NBQX;oligodendrocytes; excitotoxicity

Differential regional vulnerability to hypoxic/ischemic brain injurymay be dependent on the maturational stage of the neuronal andnon-neuronal cells in a given region. A common example of suchage-dependent regional susceptibility is the exclusively white mat-ter injury seen in infants as a complication of premature birth,referred to as periventricular leukomalacia (PVL). PVL is theprincipal neuropathological correlate of cerebral palsy. The lesionis defined by focal necrosis of the deep periventricular white matterinvolving all cellular components, combined with a more diffusewhite matter injury that appears selective for developing oligoden-drocytes (OLs) (Gilles and Averill, 1977; Dambska et al., 1989;Rorke, 1998). Reduced cerebral myelin is the most prominentsubsequent cerebral abnormality observed in premature infantswith evidence of PVL in the neonatal period (Paneth et al., 1990;Rorke, 1992; Iida et al., 1995; Olsen et al., 1997; Skranes et al.,1997; Inder et al., 1999).

A propensity to cerebral ischemia caused by impaired cerebro-vascular autoregulation, combined with a selective vulnerability ofimmature OLs to ischemic injury (Volpe, 1997), may contribute tothe prevalence of this lesion in the preterm infant. Developing OLsin vitro have been demonstrated to be more vulnerable than aremature, myelin basic protein (MBP)-expressing OLs to oxidativestress (Back et al., 1998) and to glutamate receptor (GluR)-mediated ischemic death (Fern and Moller, 2000). OLs appear tobe more vulnerable than are other glia when exposed to hypoxia/hypoglycemia in vitro (Lyons and Kettenmann, 1998). Furthermore,a number of in vivo studies have demonstrated selective whitematter injury after experimental hypoxia/ischemia in the rat brainduring early postnatal development (Rice et al., 1981; Towfighi etal., 1991; Sheldon et al., 1996; Yue et al., 1997; Ikeda et al., 1998;Reddy et al., 1998; Matsuda et al., 1999).

Both clinical and experimental studies indicate that hypoxia/ischemia is a major underlying cause of PVL. Experimental mod-els of ischemia in immature animals implicate glutamate as acritical factor in the pathogenesis of brain injury. Hypoxic/ischemicconditions result in elevated cerebral glutamate levels in the im-mature rat brain, measured by in vivo microdialysis (Benveniste etal., 1984; Silverstein et al., 1991). Clinical relevance of the exper-imental studies is suggested by the demonstration of elevatedglutamate in the CSF of term infants after perinatal hypoxia/ischemia (Hagberg, 1992). Glutamate has been shown to be toxic tooligodendroglia in vivo and in vitro by receptor-independent (Okaet al., 1993; Yoshioka et al., 1996; Back et al., 1998) and receptor-mediated mechanisms (Yoshioka et al., 1995, 1996; Matute et al.,1997; McDonald et al., 1998; Pitt et al., 2000). OLs express func-tional GluRs in vitro, and these are exclusively of the non-NMDAsubtype (Gallo et al., 1994; Patneau et al., 1994).

The purpose of this study was to examine in vivo the contributionof GluR-mediated toxicity to the selective loss of immature OLs inage-dependent cerebral white matter injury. First, we evaluated thesensitivity of immature white matter to experimental hypoxia/ischemia at the age when the cerebral white matter of a rat isprimarily populated by immature OLs. To establish whether thisinjury involved GluR activation, we confirmed the presence ofAMPA GluRs on the vulnerable cells and then assessed the pro-tective efficacy of treatment with the non-NMDA antagonist6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX). Wefurther tested the white matter selectivity and age-dependent na-ture of the GluR-mediated injury with glutamate agonist injectionsat different ages.

MATERIALS AND METHODSSubjects. Litters of male Long–Evans rat pups (Charles River Laborato-ries, Wilmington, MA) were raised with dams in a temperature-controlledenvironment with 12 hr light /dark cycles. All procedures were approvedand in accordance with guidelines set by the institutional animal care anduse committee. Pups underwent intracerebral injections or carotid ligationand hypoxia at ages postnatal day 4 (P4), P7–P8, and P10–P11. They wereallowed to recover on a thermal blanket at 33–34°C (baseline temperaturefor P7–P10 rats) and returned to their dam for 48–96 hr before beingkilled.

Normal development of oligodendrocytes in vivo. We evaluated changes inmyelin expression with age to assess the critical ages of OL development inthe neonatal rat. The corpus callosum and cortical and subcortical whitematter at the level of the dorsal hippocampus were evaluated by immuno-

Received July 7, 2000; revised Sept. 20, 2000; accepted Oct. 2, 2000.This work was supported by National Institutes of Health–National Institute of

Neurological Disorders and Stroke Grants NS 38475 (J.J.V. and F.E.J.), HD 18655(J.J.V.), and NS 31718 (F.E.J.); National Institutes of Health–National Institute ofChild Health and Human Development Grant HD 01359; and a grant from the HearstFoundation (P.L.F.). We thank S. Koh and J. Fu for technical assistance and H.Kinney for discussions.

Correspondence should be addressed to Dr. Frances E. Jensen, Enders 348, De-partment of Neurology, Children’s Hospital, 300 Longwood Avenue, Boston, MA02115. E-mail: [email protected] © 2000 Society for Neuroscience 0270-6474/00/209235-07$15.00/0

The Journal of Neuroscience, December 15, 2000, 20(24):9235–9241

cytochemistry for MBP in rats at P4, P7, P9, P11, and P18. Consistent withthe results of others (Hardy and Reynolds, 1991; Back et al., 1998), ourevaluation showed that MBP progressively increased during the first 3weeks of postnatal life (see Fig. 1). No MBP staining was demonstrated atP4/5 (n 5 6). At P7 (n 5 6) occasional cell bodies with limited processesexpressed MBP, and these were generally present in the basal ganglia andlateral internal capsule. By P11 (n 5 6) MBP staining is heavy throughoutthe corpus callosum and internal and external capsule, with MBP-positiveprocesses extending into the overlying cortex. By P18 (n 5 4) MBPexpression was abundant in white matter tracts and throughout the cortex.Immature OLs, represented by their expression of the O1 surface marker,predominate before P11 (Gard and Pfeiffer, 1990). At P4, OL progenitorsexpressing O4 surface antigen, but not O1, populate the white matter. ByP7 white matter is populated primarily with immature OLs expressingboth O4 and O1 antigen (results not shown).

Carotid ligation with hypoxia. Selective white matter injury was producedin P7 rats by unilateral carotid ligation followed by hypoxia (6% O2 for 1hr), modified from the method of Jensen et al. (1994). Rats were anesthe-tized with ether, and the proximal internal carotid artery was isolated fromthe sympathetic chain, clamped, and cauterized. The neck wound wasclosed, and the animals were allowed to recover for 1 hr on a thermalblanket, maintaining body temperature at 33–34°C. The rats were thenplaced in a sealed chamber infused with nitrogen to a level of 6% O2, alsoon a thermal blanket maintaining body temperature at 33–34°C throughouthypoxia. Body temperature was monitored by rectal probe before and aftersurgery and hypoxia and did not differ between groups. After a 1–2 hrperiod of recovery, the rats were returned to their dam. Rats were killed 96hr after injection and brains were perfused with 4% paraformaldehyde,post-fixed for 1 hr, and then cryoprotected in 30% sucrose in PBS. Todetermine the protective effect of GluR blockade in hypoxic /ischemicwhite matter injury, rats were randomized for treatment with NBQX (20mg/kg; n 5 7) or vehicle (sterile water; n 5 9) immediately after removalfrom hypoxia; a repeat treatment was given every 12 hr for 48 hr.

Stereotactic intracerebral injections. To evaluate the susceptibility of im-mature OLs to GluR-mediated toxicity in vivo, we performed stereotactic,intracerebral injections of AMPA into the pericallosal white matter ofdeveloping rats of several ages, on the basis of a modification of thetechnique described by McDonald et al. (1998). Rats were anesthetized byether inhalation, and temperature was maintained at 33–34°C throughout,monitored by a rectal probe. By the use of aseptic surgical technique, ascalp incision was made in the skull surface, and a burr hole was placedabove the desired injection location. The rat was placed in a stereotacticapparatus, and a pulled-glass micropipette attached to a nanoinjector(Drummond Scientific) was lowered with a micromanipulator to the peri-callosal white matter, 1 mm lateral to the midline and 1 mm posterior tobregma. Five nanomoles of AMPA combined with 5 nmol of MK-801 in a0.5 ml volume were injected in the experimental groups (P4, n 5 4; P7, n 58; P11, n 5 6), and 5 nmol of MK-801 alone in an equivalent volume wasinjected in the control group (P7, n 5 8). Average weights of each groupare as follows: P4, 8.3 6 0.6 gm; P7, 12.2 6 3.3 gm; and P11, 28.5 6 1.6 gm.After injection, the scalp wound was sutured closed, and the pup recoveredon a heated blanket to maintain temperature at 33–34°C and then wasreturned to its dam. Rats were killed 48–72 hr after injection and brainswere perfused with 4% paraformaldehyde, post-fixed overnight, and cryo-protected in 30% sucrose solution in PBS.

To determine the effect of GluR blockade on white matter injury, ratswere evaluated using systemic treatment with the AMPA antagonistNBQX (20 mg/kg, i.p.; every 12 hr for 48 hr), with the first dose given 30

min before the intracerebral injection. A P7 litter was randomized fortreatment with intraperitoneal NBQX or an equivalent volume of sterilewater (vehicle).

Histolog ical analysis and immunochemistry. Developmental studies ofMBP and GluRs were performed on 50 mm floating sections. For GluRanalysis, sections were incubated in 10% normal goat serum in PBS toblock nonspecific binding and then in O4 or O1 monoclonal antibody at1:800 in 5% normal goat serum. Primary antibody was labeled withfluorescein-tagged anti-mouse IgM antibody (Vector Laboratories, Burlin-game, CA). For double-labeling, sections were then permeabilized with0.1% Triton X-100 in 5% normal goat serum in PBS, incubated overnightin anti-GluR4 (5 mg/ml; Chemicon, Temecula, CA), and stained withbiotinylated anti-rabbit IgG followed by an avidin–Texas Red conjugate.

For all experimental rats, serial 20 mm coronal sections were cut bycryostat from the anterior extent of the lateral ventricles through theposterior extent of the dorsal hippocampus. Representative sections werestained with hematoxylin and eosin (HE) for routine evaluation and within situ end-labeling (ISEL) for detection of DNA fragmentation as asensitive method for evidence of cell death (Wijsman et al., 1993).Mounted sections were treated with pronase (1 gm/ml; Boehringer Mann-heim, Indianapolis, IN), rinsed in 2% glycine and then H2O, and incubatedfor 1 hr at room temperature with 50 mg/ml DNA polymerase I (Promega,Madison, WI) and 10 mM each biotin-21-dUTP (Clontech, Cambridge,UK), dCTP, dATP, and dGTP dissolved in buffer (50 mM Tris-HCl, 5 mMMgCl2, 10 mM b-mercaptoethanol, and 0.005% BSA). Biotin end-labeledDNA fragments were detected using avidin–biotin-peroxidase complexamplification (Vectastain Elite; Vector Laboratories) with diaminobenzi-dine tetrahydrochloride detection.

For MBP evaluation, adjacent mounted sections were incubated in5–10% normal goat serum for 1 hr to block nonspecific binding andconcurrently permeabilized in 0.1% Triton X-100. Slides were incubatedwith MBP antibody (SMA-99; Sternberger Monoclonals, Baltimore, MD)at a dilution of 1:800 in PBS with 1% normal goat serum plus 0.1% TritonX-100 overnight at 4°C, followed by incubation with Oregon Green anti-mouse IgG antibody (Molecular Probes, Eugene, OR). For detection ofimmature OLs, mounted sections were blocked in 10% normal goat serumfor 1 hr, incubated overnight at 4°C in O1 monoclonal antibody at adilution of 1:800 in PBS with 5% normal goat serum, rinsed, and incubated1 hr in Texas Red anti-mouse IgM antibody (Vector Laboratories).

Assessment of lesion size and statistical analysis. Data for AMPA injectionexperiments were collected from coronal sections stained with HE. Serialsections were evaluated for the extent of white matter and neuronal injuryby comparison with the contralateral side (Towfighi et al., 1994). Whitematter injury was graded by a blinded observer on a 0–3 scoring scale asfollows: 0, no discernable injury; 1, injury limited to the immediate area ofthe injection site; 2, more severe injury ,1 mm anterior–posterior (AP)and limited to the pericallosal region; and 3, injury extending .1 mm APand extending away from the pericallosal region into distal white matter.The mean severity at each age was compared by one-way ANOVA. Theinjury resulting from AMPA injection at P7 was compared against controland against NBQX-treated animals by two-tailed t tests assuming unequalvariances.

Coronal sections stained with HE were assessed histologically for cor-tical injury by light microscopy. Adjacent serial sections were also stainedwith OL-specific markers of immature and mature OLs and used tocompare the extent of white matter depletion after hypoxia/ischemia.Three adjacent pairs of coronal sections were evaluated for each rat, at thelevel of the anterior hippocampus, mid-dorsal hippocampus, and posterior

Figure 1. Developmental expression of MBP-positive cells in pericallosal white matter of im-mature rat brain. A, B, Lack of mature MBP-producing OLs at P4 (A) and the initialappearance of sparse MBP expression at P7 (B).C, D, Rapid progression to numerous MBP-producing OLs by P11 (C) and a less dense butmature-appearing pattern at P18 (D). Scale bar,100 mm.

9236 J. Neurosci., December 15, 2000, 20(24):9235–9241 Follett et al. • NBQX Attenuates Excitotoxicity in White Matter

dorsal hippocampus, by immunocytochemistry with OL-specific antibodiesfor O1 and MBP. The white matter staining was compared ipsilateral andcontralateral with the ligation, and lesion severity was assigned a value ona scale of 0–3 as follows: 0, the ipsilateral and contralateral hemispheresare similar; 1, change ipsilateral to the ligation is limited to a loss ofstaining in the cortical processes; 2, loss of staining includes thinning of theperiventricular white matter; and 3, thinning of the white matter tractsincludes a full thickness loss of staining in the capsule. A mean severityscore was obtained for immature and mature OL markers in each rat. Thelesion severity for each marker was compared statistically between thegroup treated with vehicle and the group treated with NBQX with atwo-tailed t test.

RESULTSSelective vulnerability of immature white matter tohypoxic ischemiaImmunocytochemistry revealed little MBP expression before P7; aprogressive increase between P7 and P18 indicates that whitematter is predominantly populated with immature OLs at P7 (Fig.1). Because the white matter of premature infants at high risk forhypoxic/ischemic white matter injury is also populated with imma-ture OLs (Kinney and Back, 1998), we chose this age to deliver thehypoxic/ischemic insult. Unilateral carotid ligation followed byhypoxia (6% O2 for 1 hr) at P7 resulted in a reproducible andregionally specific injury in the periventricular and subcortical

white matter (Fig. 2A). Injury was limited to the white matterwithout evidence of injury to cortical neurons. Histological obser-vation at 48 hr after hypoxia/ischemia demonstrated numerousISEL positively stained cells within the subcortical white matteripsilateral to the ligation (Fig. 2B), but not within ipsilateral over-lying cortex. Immunostaining of sections from rats killed 96 hr afterhypoxia/ischemia demonstrated diminished expression of the O1marker for immature OLs in seven of nine rats in subcortical whitematter ipsilateral to the carotid ligation when compared with ex-pression in the contralateral hemisphere (Fig. 3). Ipsilateral injuryincluded the loss of MBP expression in the OL processes extendinginto the cortex and decreased thickness of the periventricular whitematter and external capsule in four of nine rats. All rats showed anipsilateral decrease in the presence of MBP in the OL processesextending into the cortex (Fig. 4A,B). In summary, hypoxic/isch-emic injury at P7 resulted in selective white matter injury asdemonstrated by the loss of MBP expression in OL processes 96 hrlater.

Figure 2. Selective white matter injury follows hypoxia/ischemia to imma-ture rat brain at P7. A, Coronal section through the dorsal hemisphere of arat killed 48 hr after carotid ligation and hypoxia at P7, demonstrating theabsence of injury to the overlying cortex. The arrow points to tissue loss inpericallosal white matter. Top of figure represents cortical surface. B,High-power view of dying cells in an adjacent coronal section. ISEL-positive cells are common in pericallosal white matter 48 hr after hypoxic/ischemic insult. Scale bars: A, 100 mm; B, 10 mm.

Figure 3. Loss of immature OLs in subcortical white matter after hypoxia/ischemia. A, B, O1 staining of subcortical white matter tracts in coronalsections of a P11 rat. Sections are contralateral (A) and ipsilateral (B) to theunilateral carotid ligation that was followed by hypoxia at P7. Arrows pointto an area of much reduced O1 staining after hypoxia/ischemia. Scale bar,100 mm.

Figure 4. Effect of NBQX on MBP ex-pression in cerebral white matter afterhypoxia/ischemia. A–D, MBP expressionin the subcortical white matter of a P11 ratafter unilateral carotid ligation and hyp-oxia at P7, with and without NBQX treat-ment. MBP staining of white matter tractscontralateral (A) and ipsilateral (B) to theligation in a vehicle-treated control andcontralateral (C) and ipsilateral (D) tothe ligation in a littermate post-treatedwith NBQX demonstrates significant at-tenuation of myelin loss with treatment.Scale bar, 100 mm.

Follett et al. • NBQX Attenuates Excitotoxicity in White Matter J. Neurosci., December 15, 2000, 20(24):9235–9241 9237

Presence of AMPA-preferring GluRs in OLs at P7Because of the potential role of glutamate in hypoxic/ischemicwhite matter injury and the presence of AMPA-preferring GluRson OLs in vitro, we evaluated whether AMPA-preferring receptorswere present on immature OLs at this age in vivo. Immunocyto-chemical analysis using AMPA receptor subunit antibodies dem-onstrated robust expression of the GluR4 subunit in white matter atP7 (n 5 4). Immunocytochemistry with the O1 antibody to detectimmature OLs (the primary OL stage present at P7) and theGluR4 antibody demonstrated widespread double-labeling in thecorpus callosum, pericallosal white matter, and external and inter-nal capsule (Fig. 5). In contrast, little coexpression of GluR4 wasdetected in the predominant OL stages seen at younger (P4;O41O12) and older (P11; MBP1) ages. These data confirm therelative high expression of AMPA receptors in immature OLs incerebral white matter at this vulnerable age.

Systemic NBQX attenuates hypoxic/ischemic whitematter injuryBecause of the presence of AMPA receptors on immature OLsduring the time period of susceptibility to hypoxia/ischemia, weexamined whether AMPA receptor blockade with NBQX wouldattenuate the injury. Pups treated with NBQX (n 5 7) at thetermination of the period of hypoxia after carotid ligation show amarked attenuation of the ipsilateral decrease in O1 and MBPstaining seen 96 hr after the insult (Fig. 4) when compared withuntreated littermate controls (n 5 9). A semiquantitative analysisof lesion severity demonstrated significant attenuation of whitematter injury in rats post-treated with NBQX, compared withvehicle-treated controls, when evaluated for either O1 expression( p , 0.005) or MBP expression ( p , 0.001) (Fig. 6). Treated pupsshowed either no detectable injury (three of seven pups) or mild

ipsilateral injury, generally limited to slight loss of MBP or O1expression in the cortical processes.

Systemic NBQX attenuates AMPA-mediated whitematter injuryNBQX attenuation of the selective white matter injury after hyp-oxia/ischemia implicates GluR-mediated toxicity as an importantmechanism of injury in immature OLs. To confirm a relationshipbetween the activation of AMPA receptors in cerebral white mat-ter and the vulnerability to injury, we injected AMPA directly intoimmature white matter. Intracerebral injections of 5 nmol ofAMPA plus 5 nmol of MK-801 produced white matter injury in P7rat pups (n 5 8) in the absence of significant cortical or hippocam-pal injury. Most P7 rats demonstrated areas of hypercellularitysurrounding tissue disruption and necrosis in the pericallosal whitematter, frequently with hemorrhage and with little cortical injury.Minimal to no injury was present in vehicle-injected controls (MK-801 alone; n 5 7) at the same age (t test, p , 0.001). The severityof white matter injury was evaluated by the size of the resultinglesion (Fig. 7). Staining with ISEL showed evidence of cell death inthe white matter of the pericallosal region at P7 in rats injectedwith AMPA and of injury limited to the site of the needle track incontrols. Systemic administration of the AMPA receptor antago-nist NBQX significantly attenuated white matter injury at P7 (Fig.7A, t test, p , 0.005). These results suggest a receptor-mediatedmechanism of injury from AMPA injections and confirm the effi-cacy of systemic NBQX, administered as a post-treatment.

Age-dependent vulnerability of cerebral white matterto AMPATo determine whether AMPA toxicity was age dependent, wecompared lesion size after intracerebral AMPA injections at P4,

Figure 5. Immature OLs expressing O1antibody double-label for non-NMDAGluRs in vivo at P7. D, E, O1-expressingimmature OLs in the periventricularwhite matter of a P7 rat (D) and GluR4subunit expression in the same region (E).F, Superimposed images demonstratingthe colocalization of receptor protein withOLs at P7. Arrows point to individualO11 immature OLs also expressingGluR4. A, B, O4-expressing OL progeni-tors ( A) and GluR4 subunit expression(B) in the periventricular white matter ofa P4 rat. G, H, MBP-expressing OLs (G)and GluR4 subunit expression (H ) in theperiventricular white matter of a P11 rat.C, I, Superimposed images at P4 (C) andP11 ( I ) showing the absence of colocal-ization at the younger and older ages.Scale bar, 50 mm.


P7, and P11. Injections at both P4 (n 5 4) and P11 (n 5 6)produced significantly less white matter injury than did injections atP7 (one-way ANOVA, p , 0.001). Comparison of the resultingwhite matter injury is demonstrated by a histogram of the perical-losal lesion size (Fig. 7B). However, whereas pups injected at P4had minimal cortical injury, animals injected at P11 exhibitedwidespread injury in the overlying cortex and adjacent hippocam-pus. These results indicate that intracerebral injections of AMPAproduce an in vivo white matter lesion in an age-dependent man-ner; the most severe and specific lesion is at P7, and less severe andless specific lesions are at both younger (P4) and older (P11) ages.

DISCUSSIONThis study suggests that the age-dependent vulnerability of OLs tohypoxic/ischemic injury may be mediated by GluR activation andmay be correlated with maturational differences in GluR expres-sion. We demonstrated an increased vulnerability of white matterto hypoxic/ischemic injury at P7, a maturational stage when whitematter is populated with immature OLs. We confirmed the pres-ence of AMPA-preferring GluR subunits on the immature OLs atthis age in vivo. After hypoxia/ischemia at P7, systemic treatmentwith the AMPA receptor antagonist NBQX significantly attenu-ated the selective white matter injury. Furthermore, the vulnera-bility of white matter to intracerebral injections of AMPA appearsto be age dependent, with the greatest susceptibility to injury at P7.Hypoxic/ischemic and AMPA-induced injury are each blocked byNBQX, indicating that the toxicity is receptor mediated. Theseresults suggest that GluR-mediated toxicity is a contributing factorin the age-dependent, selective injury to developing OLs afterhypoxia/ischemia in the immature brain.

The progressive development and differentiation of oligodendro-cytes from progenitors to mature, myelinating oligodendrocyteshas been well characterized both in vitro (Gard and Pfeiffer, 1990;Asou et al., 1995) and in vivo (LeVine and Goldman, 1988; Hardyand Reynolds, 1991, 1997). Similar developmental sequences of OLmaturation are observed in the white matter of the rat and human(Kinney and Back, 1998), further supporting the use of the rat as anexperimental model of PVL. Expression of MBP does not beginuntil P7, and this expression is followed by a rapid increase inmyelin over the following few days. Therefore, this developmentalstage correlates with a time in premature infants when white matteris highly vulnerable to injury.

GluR expression also appears to be maturation dependent. Thepresence of AMPA-preferring GluRs on OLs is well established

(Gallo et al., 1994; Meucci et al., 1996; Matute et al., 1997), and thevariable expression of AMPA receptor subtypes in different brainregions during development has been demonstrated by in situhybridization (Pellegrini-Giampietro et al., 1991). In agreementwith these results, we demonstrate relatively high levels of expres-sion of AMPA receptors on immature OLs in vivo at P7, in areasvulnerable to hypoxic/ischemic injury. This age is before the timein development when expression rapidly increases in the cortex(Pellegrini-Giampietro et al., 1991; Petralia and Wenthold, 1992).The presence of GluRs on immature OLs, at an age when there isa comparative lack of expression in the cortex, may explain therelatively specific vulnerability of white matter at this maturationalstage to GluR-mediated toxicity.

Moderate hypoxia/ischemia in P7 rats results in a selective whitematter injury with relative cortical sparing. The proportion ofcortical injury after hypoxia/ischemia varies with age; white matterinjury is more common after cerebral hypoxia/ischemia in imma-ture rats, whereas cortical and subcortical gray matter infarction istypically seen in the adult (Rice et al., 1981; Andine et al., 1990;Sheldon et al., 1996; Uehara et al., 1999). In addition, selectivewhite matter injury attributable to AMPA injections is age depen-dent. Intracerebral AMPA caused the most selective and severewhite matter injury at P7, with younger ages less prone to injuryand older ages more susceptible to neuronal injury. White matterinjury is not seen with MK-801 injections at P7, confirming thatthis was not a mechanical injury. Therefore, there is an age-dependent injury to white matter resulting from either hypoxia/ischemia or GluR agonist injections.

The AMPA antagonist NBQX was effective at attenuating im-mature white matter injury in vivo, caused either by direct receptoractivation or by hypoxia/ischemia. NBQX blocked the injury at P7caused by AMPA injections, consistent with the results of others(Yoshioka et al., 1996; McDonald et al., 1998) and suggesting areceptor-mediated cause of injury. However, in addition to block-ing activation of non-NMDA receptors, NBQX has been demon-strated to induce a protective hypothermia (Young et al., 1983;Nurse and Corbett, 1996). We observed no difference in rectaltemperatures between the treated and untreated groups, consistentwith reports of others (Hagberg et al., 1994). A protective effect ofhypothermia in P7 rats is significant only if present during hypoxia(Yager et al., 1993), and in the current study NBQX treatment wasinitiated after hypoxia. The presence of GluRs in the vulnerablecells and the attenuation of hypoxic/ischemic white matter injury

Figure 6. Evaluation of white matter injury with the OL-specific markersO1 and MBP. Comparison of the severity of white matter injury 96 hr afterhypoxia/ischemia at P7 in treated (n 5 7) and untreated (n 5 9) pups showssignificant attenuation of injury with NBQX post-treatment (20 mg/kg, i.p.;every 12 hr for 48 hr). NBQX treatment attenuates the loss of O11 OLs (A;*p , 0.005) and MBP expression (B; *p , 0.001) ipsilateral to the ligation.CTL, Control.

Figure 7. White matter injury after intracerebral injections of AMPA. A,Injury after injection with 5 mol AMPA plus 5 nmol MK-801 (n 5 8) wassignificantly more severe than with MK-801 alone (control, CTL; n 5 7; **p, 0.001) B, The effect on rats of different ages. Significantly greater injuryseverity is shown in white matter at P7 (n 5 8; one-way ANOVA; *p ,0.001) compared with that at younger (P4; n 5 4) and older (P11; n 5 6)ages and with MK-801 alone at P7 (n 5 7).


with AMPA receptor blockade together implicate receptor-mediated excitotoxicity as a contributing factor in hypoxic/ischemicwhite matter injury in the immature brain.

The in vivo efficacy of NBQX is consistent with previous studiesdemonstrating attenuation of GluR-mediated excitotoxicity in vitro(Yoshioka et al., 1995, 1996; Matute et al., 1997; McDonald et al.,1998; Pitt et al., 2000). In vitro, immature OLs appear to be moresusceptible to excitotoxicity than are mature OLs (Fern andMoller, 2000), and the mechanism of excitotoxicity may be calciumdependent (David et al., 1996; Yoshioka et al., 1996; Brorson et al.,1999; Li and Stys, 2000). Neither the source of elevated glutamatenor the mechanism of cell death in vivo is known; however, reverseglutamate transport from axons (Li et al., 1999; Rossi et al., 2000)or glia (Fern and Moller, 2000) has been suggested as a source. Theprotective efficacy of non-NMDA receptor blockade supports thehypothesis that selective injury to immature OLs by a receptor-mediated mechanism is sufficient to cause cerebral white matterinjury in vivo.

Our results indicate that developing white matter exhibits an agewindow of enhanced susceptibility to GluR-mediated excitotoxic-ity. Injury because of hypoxia/ischemia parallels that caused bydirect AMPA toxicity, with maximum selective white matter injuryat P7. Cerebral white matter at this age is populated primarily byimmature OLs that possess AMPA receptor subunits. In agree-ment with the proposed vulnerability of immature OLs to excito-toxicity by a GluR-mediated mechanism, the AMPA receptorantagonist NBQX attenuated both the AMPA-induced lesions aswell as the hypoxic/ischemic white matter injury. Taken together,these results suggest that hypoxic/ischemic injury in developingwhite matter is mediated at least in part by excitotoxicity viaglutamate receptors on immature OLs.

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nbqx attenuates excitotoxic injury in developing white matter

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